CN108753832A - 一种利用CRISPR/Cas9编辑大白猪CD163基因的方法 - Google Patents
一种利用CRISPR/Cas9编辑大白猪CD163基因的方法 Download PDFInfo
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Abstract
本发明公开了一种利用CRISPR/Cas9编辑大白猪CD163基因的方法，利用CRISPR/Cas9编辑大白猪CD163基因，破坏CD163受体胞外域SRCR5，敲除CD163基因第7个外显子DNA片段，CD163基因的第7个外显子的核苷酸序列如SEQ ID NO.1所示。采用本发明的方法，能够完全抵抗PRRSV感染，包括抗高致病性毒株HP‑PRRSV，而细胞表面CD163受体表达正常，其他生物学功能正常。
Description
技术领域
本发明涉及生物技术领域，具体来说，涉及一种利用CRISPR/Cas9编辑大白猪CD163基因的方法。
背景技术
猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome，PRRS)又称蓝耳病，是由猪繁殖与呼吸综合征病毒(Porcine reproductive andrespiratory syndrome viruse，PRRSV)引起，PRRS主要引起妊娠母猪流产、死胎、木乃伊胎、弱仔及各年龄阶段猪特别是仔猪呼吸道症状，特征性病变为间质性肺炎，死亡率极高，是一种高度接触性的全球性的重要传染病。2006年，我国爆发了猪高热病，对养猪业造成严重的经济损失，后来将此毒株定义为高致病型毒株(Highly pathogenic porcinereproductive and respiratory syndrome viruse，HP-PRRSV)，目前，高致病性蓝耳病(Highly pathogenic porcine reproductive and respiratory syndrome，HP-PRRS)是对养猪业损害最大的疾病之一，由于该病毒具有免疫抑制性、抗体依赖增强性、持续性感染、病毒血症维持时间长等特点，目前尚没有很好的疫苗和药物可以防控蓝耳病。
CD163是单核细胞、猪肺泡巨噬细胞(Porcine alveolar macrophage,PAMs)表面特异表达的富含半胱氨酸清道夫受体(cysteine-rich scavenger receptor,SRCR)，一般可作为PAMs细胞Marker。CD163共含有9个SRCR结构域，其中SRCR5在病毒感染过程中发挥主要的作用，有研究表明，SRCR5的缺失或破坏能抑制PRRSV的感染。同时有体外实验证明，SRCR5主要由CD163基因的外显子7负责编码。该发现为构建抗PRRSV的CD163基因编辑动物提供了依据，即通过基因编辑技术结合体细胞核移植的方法，制备出SRCR5被破坏的抗PRRSV的CD163基因编辑动物。有研究报道CD163受体敲除猪可以完全抵抗PRRSV感染，没有任何如发热、呼吸困难等症状，体内检测不到任何PRRSV抗原和抗体，组织切片显示肺部没有被PRRSV感染。
Cas9和gRNA是CRISPR/Cas9系统的基本成分，gRNA用于特异位点识别，Cas9用于切割靶位点DNA。与传统的基因组编辑技术相比，CRISPR/Cas9系统的构建更加简便、快速、廉价。研究发现，当gRNA的靶位点位于同一条染色体上时，利用Cas9和多条gRNA共转细胞，可以产生两条gRNA靶位点之间DNA片段的删除，DNA片段删除能更有效地敲除目的基因。
发明内容
本发明的目的是针对以上要解决的技术问题，提供一种能够有效敲除目的基因的利用CRISPR/Cas9编辑大白猪CD163基因的方法。
为了解决上述问题，本发明提供了一种利用CRISPR/Cas9编辑大白猪CD163基因的方法，其利用CRISPR/Cas9编辑大白猪CD163基因，破坏CD163受体胞外域SRCR5，敲除CD163基因第7个外显子DNA片段，所述CD163基因的第7个外显子的核苷酸序列如SEQ ID NO.1所示。
根据本发明提供的方法，优选地，用于敲除所述CD163基因的所述第7个外显子的gRNA的核苷酸序列如SEQ ID NO.2和SEQ ID NO.3所示。
所述蓝耳病毒包括经典毒株和高致病性毒株。具体优选地，所述高致病性毒株为高致病性蓝耳病病毒HP-PRRSV。
根据本发明的方法，通过CRISPR/Cas9进行基因编辑的步骤如下：
将核苷酸序列如SEQ ID NO.2所示的gRNA-10构建到能表达Cas9蛋白的pX458载体上，得到pX458-gRNA-10；将核苷酸序列如SEQ ID NO.3所示的gRNA-134构建到能表达Cas9蛋白的pX458R载体上，得到pX458R-gRNA-134，其包括两种能够特异性识别CD163基因并对识别位点进行打靶的CRISPR/Cas9系统；将所述pX458-gRNA-10和所述pX458R-gRNA-134共转染猪的离体胎儿肾细胞，分别打靶CD163基因上相应gRNA所识别的位点，从而删除所述CD163基因上两条gRNA所识别位点的中间序列，实现所述CD163基因第7个外显子的DNA片段的精确删除。
本发明还提供了一种利用CRISPR/Cas9编辑CD163基因制备抗蓝耳病大白猪的方法，其利用CRISPR/Cas9编辑大白猪CD163基因，破坏CD163受体胞外域SRCR5，敲除CD163基因第7个外显子DNA片段，CD163基因的第7个外显子的核苷酸序列如SEQ ID NO.1所示。优选地，用于敲除所述CD163基因的所述第7个外显子的gRNA的核苷酸序列如SEQ ID NO.2和SEQID NO.3所示。
本发明还提供了一种研究CD163基因在大白猪蓝耳病毒感染过程中的作用的方法。
本发明首次通过CRISPR/Cas9基因编辑技术敲除猪肺泡巨噬细胞表面CD163受体胞外域SRCR5，培育抗蓝耳病大白猪；本发明培育的抗蓝耳病大白猪只敲除了PRRSV侵入猪肺泡巨噬细胞必需的CD163受体胞外域SRCR5，而不是敲除整个CD163基因，即CD163基因仍然在猪肺泡巨噬细胞膜上表达，CD163受体其他生物学功能仍然保存。
为了确证本发明培育的抗蓝耳病大白猪能够完全抵抗HP-PRRSV感染，通过活体攻毒试验，并选择毒力极强的JXA1、MYUAN两种毒株以3×105TCID50分别攻毒42天。结果显示，基因编辑猪血清蓝耳抗原抗体双阴性，解剖后肺部无病变，表明基因编辑猪完全不感染毒力极强的JXA1、MYUAN毒株。故以上试验确证本发明培育的抗蓝耳病大白猪能够完全抵抗HP-PRRSV感染。
另外，本发明培育的抗蓝耳病大白猪有3种基因型：1)DEL/DEL：CD163受体SRCR5双等位基因精准删除纯合子；2)DEL/KO：CD163受体SRCR5精准删除，另一个等位基因CD163敲除(不只删除CD163受体SRCR5)；3)KO/KO：两个等位基因CD163敲除(不只删除CD163受体SRCR5)。以上3种基因型均能完全抵抗蓝耳病毒感染。
因此，本发明培育的抗蓝耳病毒大白猪可以作为PRRSV病毒研究模型，包括PRRSV致病机理研究、挖掘新型抗病或易感基因研究；对研究PRRSV致病机理及挖掘新型抗病或易感基因具有重要意义，为PRRSV研究提供一个新的模型。
附图说明
图1为CRISPR/Cas9编辑大白猪CD163基因示意图。
图2为猪胎儿肾细胞中gRNA-10和gRNA-134的删除效率鉴定结果。
图3为JXA1毒株攻毒42天后，野生型大白猪与基因编辑大白猪存活统计结果。
图4为MY毒株攻毒42天后，野生型大白猪与基因编辑大白猪存活统计结果。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
本发明以下实施例的统计学分析：所有试验至少3次独立重复，结果采用平均值和标准误表示，使用单因素方差分析和T检测分析。所有统计分析均采用以P<0.05作为具有显著统计学差异的检验标准，分析软件为SPSS 16.0和GraphPad Prism 5。
实施例1
利用CRISPR/Cas9编辑CD163基因制备基因编辑细胞
1、离体猪胎儿肾细胞的获得
猪胎儿肾细胞从大白猪胎儿肾脏中分离，在超净台内进行猪胎儿肾细胞的分离。用剪刀和镊子取下胎儿的肾脏组织，将取下的组织依次在75％(v/v)酒精以及添加了抗生素的PBS里反复清洗，用小剪刀将组织块剪至1立方毫米大小，1600rpm离心5min去除PBS，再加入带抗生素的20％FBS的DMEM，轻轻吹打均匀，放入37℃细胞培养箱培养。放入细胞培养箱后，不要挪动培养皿，三天后，可观察到猪胎儿肾细胞已爬满至整个培养皿，再进行一般传代细胞的消化培养即可。
2、含有编辑型CD163基因的细胞的获得
1)质粒转染进细胞获得CD163基因编辑细胞
针对猪CD163基因第7个外显子(SEQ ID NO.1)设计两条gRNA，分别构建到能表达Cas9蛋白的pX458和pX458R载体上，形成两种能够特异性识别CD163基因并对识别位点进行打靶的CRISPR/Cas9系统(见图1)，即pX458-gRNA-10和pX458R-gRNA-134。
设计用于编辑猪CD163基因的两条gRNA序列如下：
gRNA-10：5’-ggaaacccaggctggttgga-3’(SEQ ID NO.2)；
gRNA-134：5’-ggaactacagtgcggcactg-3’(SEQ ID NO.3)。
采用电转的方法将5ug pX458-gRNA-10和5ug pX458R-gRNA-134共转染1*106猪胎儿肾细胞，得到CD163基因编辑细胞。电转严格按照试剂盒和电转仪说明书操作。
2)鉴定含有编辑型CD163基因的细胞
设计用于扩增删除区域的引物对如下：
CD163-DF3：5’-ctgctcagcccacaggaaac-3’(SEQ ID NO.4)；
CD163-DR3：5’-gccattcaccaagcggattt-3’(SEQ ID NO.5)。
将上述1)得到的编辑细胞基因组DNA作为模板，用CD163-DF3与CD163-DR3组成的引物对进行PCR扩增。
如图2所示，分别回收约441bp(野生型条带大小)和317bp(删除目标区域后的条带大小)扩增产物并连接至T载体进行测序分析。计算克隆中含有编辑型CD163基因的克隆比例，即为该CRISPR/Cas9系统编辑效率，编辑效率越高，获得CD163基因编辑猪的比例越高。结果如以下表1所示。
表1
实施例2
1、体细胞核移植获得CD163基因编辑猪
从健康大白母猪体内采取挑选发育阶段适宜的卵巢，用注射器抽取卵巢表面直径在3-5mm的卵泡中的内含物，将内含物在TL-PVA中稀释并重悬形成悬浊液。将悬浊液在37℃环境下静置至卵母细胞沉淀完全，将沉淀吸出置于在体视镜下用移液器或口吸管挑选卵周细胞完整的卵母细胞。将挑选的健康卵母细胞放入含有10％(重量百分比)卵泡液、FSH、LH、EGF的TCM-199中培养22h。再用移液器或口吸管将卵母细胞移到含有10％(重量百分比)卵泡液、EGF的TCM-199中继续培养22h。经过44h培养成熟后挑选已经排出第二极体的健康成熟卵母细胞作克隆胚胎用。
将上述制备的大白猪的含有编辑型CD163基因的细胞，于5％(体积浓度)CO2、37℃饱和湿度的细胞培养箱培养，待细胞长至对数生长期时，即可用于核移植操作。
待卵母细胞体外培养成熟后，用采用电融合法将含有编辑型CD163基因的细胞群进行体细胞核移植，并在24h之内进行胚胎移植，制备不同品种的CD163基因型的基因编辑猪。
2、CD163基因编辑猪的鉴定
采取少量CD163基因编辑猪的耳组织样提取基因组作为模板，用上述CD163-DF3与CD163-DR3组成的引物对(SEQ ID NO.4和SEQ ID NO.5)进行PCR扩增，并克隆测序，鉴定克隆猪的基因型，结果如以下表2所示。
表2
实施例3：抗蓝耳病克隆猪活体攻毒实验
1.4-6周龄仔猪16头(8头基因编辑猪+8头野生型猪，打好耳号)分成两个攻毒室(用于两种不同PRRSV毒株(JXA1毒株和MY毒株)攻毒)，每个攻毒室各8头(4头基因编辑猪+4头野生型大白猪，8头混在一起)。
2.适应环境1周后，每头猪滴鼻攻毒剂量：3ml细胞培养液含2×105TCID50病毒。
3.攻毒后0(攻毒前当天)、4、7、14、21、28、35、42天采血、测量体温、称重、临床症状观察(呼吸、神经等症状，打分统计)，血清冻存于-80℃备用。攻毒期间，若有猪只死亡，需统计。攻毒后野生型猪与基因编辑猪精神状态进行对比：野生型猪JXA1攻毒后毛松、咳嗽、精神差，而基因编辑猪JXA1攻毒后毛色光亮、精神好。分别攻毒JXA1、MY毒株42天后，野生型猪与基因编辑猪存活统计如图3、图4所示。统计结果表明，基因编辑猪的生存率为100％，优于野生型猪。
4.42天后，安乐死剖解，病料组织10％福尔马林固定，石蜡包埋，为病理切片准备。解剖后检测指标：PRRSV病毒血症检测、PRRSV抗体检测、肺泡巨噬细胞表面CD169、CD163表达检测、CD163受体生物学功能验证、临床症状观察(呼吸、神经等症状，打分统计)、攻毒后0、4、7、14、21、28、35、42天称体重，计算平均日增重、肺组织病理切片检查和免疫组化检查。
序列表
<110> 中山大学
<120> 一种利用CRISPR/Cas9编辑大白猪CD163基因的方法
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aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 120
tgctgtgcag ggaactacag tgcggcactg tggtttccct cctgggggga gctcactttg 180
gagaaggaag tggacagatc tgggctgaag aattccagtg tgaggggcac gagtcccacc 240
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Claims (3)
1.一种利用CRISPR/Cas9编辑大白猪CD163基因的方法，其特征在于：利用CRISPR/Cas9编辑大白猪CD163基因，破坏CD163受体胞外域SRCR5，敲除所述CD163基因第7个外显子DNA片段，所述CD163基因的第7个外显子的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的方法，其特征在于：用于敲除所述CD163基因的所述第7个外显子的gRNA的核苷酸序列如SEQ ID NO.2和SEQ ID NO.3所示。
3.根据权利要求1或2所述的方法，其特征在于，通过CRISPR/Cas9进行基因编辑的步骤如下：
将核苷酸序列如SEQ ID NO.2所示的gRNA-10构建到能表达Cas9蛋白的pX458载体上，得到pX458-gRNA-10；将核苷酸序列如SEQ ID NO.3所示的gRNA-134构建到能表达Cas9蛋白的pX458R载体上，得到pX458R-gRNA-134，其包括两种能够特异性识别CD163基因并对识别位点进行打靶的CRISPR/Cas9系统；将所述pX458-gRNA-10和所述pX458R-gRNA-134共转染猪的离体胎儿肾细胞，分别打靶CD163基因上相应gRNA所识别的位点，从而删除所述CD163基因上两条gRNA所识别位点的中间序列，实现所述CD163基因第7个外显子的DNA片段的精确删除。
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